We have developed yeast vacuoles as a paradigm of membrane fusion in endocytic and exocytic traffic. Each stage of this traffic, from yeast to humans, uses similar Rab, effector complex, SNAREs, and SNARE chaperones, but studies of yeast vacuole fusion have unique technical advantages: superb genetics, genomics, and biochemistry of baker's yeast, ease of visualizing the vacuole and purifying it, and the rapid and quantitative assays which we've developed for fusion and its subreactions. In addition to studying vacuole fusion in vivo and with the purified organelle in vitro, we have developed in this funding period a reconstitution of proteoliposome (RPL) fusion with all-purified components: the 4 vacuolar SNAREs, the Ypt7p Rab GTPase, its effector complex HOPS (in which the vacuolar SM protein Vps33p is one of 6 subunits), the SNARE disassembly chaperones Sec17p/Sec18p, 9 physiological vacuolar lipids, and ATP. This system has revealed basic mechanistic features of fusion which underlie each aim of our proposal: (i) The fusion pathway proceeds through defined stages;we will use our published assays of tethering and trans-SNARE complex formation, and will exploit our fluorimetric assays of content mixing, lipid mixing, permeability, and lysis, using agents that interrupt fusion to seek hemifusion (partial lipid mixing without content mixing). (ii) Diverse vacuolar lipids are needed for rapid fusion, beyond simply forming a bilayer;we will explore which are needed for peripheral membrane protein binding, tethering, trans-SNARE complex assembly, and the lipid rearrangements of hemifusion and fusion. (iii) Fusion catalysts undergo regulated cycles: 1. HOPS and Sec17/18p synergistically support fusion as SNAREs cycle between cis-complexes, the uncomplexed state, and trans-complexes, 2. Gyp7p and Ccz1/Mon1 catalyze the Rab Ypt7p cycling between GTP- and GDP-bound states, and 3. the vacuolar kinase Yck3p and unknown phosphatase(s) regulate HOPS phosphorylation. These 3 cycles are likely related, and our unique tools will allow exploration of these relationships. Understanding the vacuolar HOPS complex, discovered and studied in this work, underlies studies of human arthrogryposis, renal dysfunction, and cholestasis syndromes, caused by mutations in the human VPS33 gene. Human HOPS has recently been shown to have a unique and central role in Marburg and Ebola virus invasion of human cells. The proposed studies are thus important for both basic and clinical science.

Public Health Relevance

Membrane fusion is essential for processes as diverse as cell growth, hormone secretion, and neurotransmission, but its mechanism is highly conserved from yeast to humans. By studying the fusion of yeast vacuoles (lysosomes) at each level, defining the needed proteins and lipids, purifying them, and reassembling them into defined reactions which bear on all facets of the mechanism, we have discovered fundamental components whose human homologs are required for viral infection (Ebola and Marburg virus) and underlie certain inherited human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM023377-38A1
Application #
8570773
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
1976-05-01
Project End
2017-06-30
Budget Start
2013-08-01
Budget End
2014-06-30
Support Year
38
Fiscal Year
2013
Total Cost
$857,265
Indirect Cost
$322,365
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Zick, Michael; Wickner, William (2016) Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement. Mol Biol Cell 27:2590-7
Zick, Michael; Orr, Amy; Schwartz, Matthew L et al. (2015) Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18. Proc Natl Acad Sci U S A 112:E2290-7
Orr, Amy; Wickner, William; Rusin, Scott F et al. (2015) Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion. Mol Biol Cell 26:305-15
Baker, Richard W; Jeffrey, Philip D; Zick, Michael et al. (2015) A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science 349:1111-4
Zick, Michael; Stroupe, Christopher; Orr, Amy et al. (2014) Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion. Elife 3:e01879
Zick, Michael; Wickner, William T (2014) A distinct tethering step is vital for vacuole membrane fusion. Elife 3:e03251
Zick, Michael; Wickner, William (2013) The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes. Mol Biol Cell 24:3746-53
Karunakaran, Vidya; Wickner, William (2013) Fusion proteins and select lipids cooperate as membrane receptors for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vam7p. J Biol Chem 288:28557-66
Xu, Hao; Wickner, William T (2012) N-terminal domain of vacuolar SNARE Vam7p promotes trans-SNARE complex assembly. Proc Natl Acad Sci U S A 109:17936-41
Zick, Michael; Wickner, William (2012) Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion. Mol Biol Cell 23:3429-37

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